Methods, Reagents and Kits for Flow Cytometric Immunophenotyping

ABSTRACT

The invention relates to the field of flow cytometry and more particularly to a panel of antibody reagents conjugated to fluorescent compounds. Provided are reagent compositions, comprising at least eight distinct fluorochrome-conjugated antibodies comprising a set of at least there identification antibodies for the identification of a leukocyte population of interest and at least four characterization antibodies for further characterization and/or classification of said leukocyte population. Also provided are kits and methods related to the reagent compositions.

The invention relates to the field of flow cytometry and moreparticularly to a panel of antibody reagents conjugated to fluorescentcompounds. They find their use in the immunophenotypic characterizationof normal, reactive, regenerating and neoplastic cells in peripheralblood (PB), bone marrow (BM), pleural effusions, ascitis, cerebrospinalfluid (CSF), vitreous humor, synovial fluid, bronchoalveolar lavage,urine, spleen, liver, lymph node, and other tissue samples. Flowcytometric immunophenotyping of normal, reactive, regenerating, andmalignant cells (in particular leukocytes) is currently being used formany applications in medicine, including immunology, hematology andoncology. Among others, said applications include monitoring of theimmune system; diagnosis and classification of primaryimmunodeficiencies; immunophenotyping of leukemias, lymphomas and plasmacell dyscrasias; monitoring of low frequencies of malignant leukocytesas measure for treatment effectiveness; diagnosis and monitoring ofclonal disorders such as paroxysmal nocturnal hemoglobinuria (PNH) andmastocytosis; evaluation of hematopoiesis or lymphopoiesis in differentclinical conditions, e.g. in healthy individuals, after stein celltransplantation or gene therapy (using hematopoietic precursor cells astarget); and evaluation of the composition and quality of cell productsto be used for therapeutic purposes.

The conventional diagnostic process in flow cytometric immunophenotypingis typically based on the usage of panels of antibodies. At presentdifferent, overlapping panels of antibodies are recommended for specificapplications. Such panels are driven either by the medical indication(e.g.: screening for cytopenias, characterization of lymphocytosis), bydisease (e.g.: acute leukemia diagnosis, lymphoma diagnosis) or diseasestatus (e.g.: diagnostic classification of ALL vs. monitoring of ALL forevaluation of treatment effectiveness). Examples of recommended antibodypanels are the European Leukemia Net (ELN) (1), the 2006 BethesdaInternational Consensus (2) panels for different subtypes ofhematological malignancies, the EGIL panels for lineage assignment andsubclassification of acute leukemias (3), the European Myeloma Network(EMN) panels for diagnosis, classification and monitoring of plasma celldyscrasias (4), the ERIC (5) and Matutes (6) score panels forimmunophenotypic subclassification of chronic lymphocytic leukemia andhairy cell leukemia vs. other chronic lymphoproliferative disorders.

These recommended antibody panels are mostly comparable betweendifferent countries and study groups, but never fully identical.Importantly, no or limited information has been provided by the variousnetworks and study groups on how the antibodies should preferably becombined into a panel of antibody reagents conjugated with specificfluorescence compounds. Only simple lists of antibodies have beenproposed, even when diagnostic laboratories can simultaneously stain asample aliquot with 2, 3, 4, 5, 6, 7, 8 or more antibodies conjugatedwith different fluorescent dyes having fluorescence emissions that canbe measured separately. Variations between centres around the world aredue to the usage of:

-   -   Comparable but not identical lists of antibodies and antibody        clones;    -   The same antibodies conjugated with different fluorochromes that        show different sensitivities. For example, an antibody reagent        might provide a negative result if the antibody is combined with        a low-sensitive fluorochrome, while a positive result could be        obtained if an antibody reagent consisting of the same antibody        combined with a different, more sensitive fluorochrome, is used        to stain the same cells.    -   Simultaneous assessment of different combinations of antibody        reagents.    -   Variable and frequently suboptimal or inappropriate strategies        for the identification of the cell population(s) of interest,        such as the CD45 gating in acute myeloblastic leukemias.

As a consequence, the seemingly comparable antibody panels do not resultin comparable diagnosis of clinical samples. In fact, thereproducibility between diagnostic laboratories is not more than 70%.Unfortunately, diagnoses are missed because of inappropriatediscrimination between normal and malignant cells or regenerating bonemarrow is erroneously diagnosed as recurrence of acute leukemia.

In U.S. Pat. No. 5,047,321, Loken and Terstappen disclosed a procedurefor the multiparameter analysis of cellular components in PB and BM.With the procedure described, these inventors could distinguish severalcellular components of PB and BM, count the number of cells within eachcomponent, and provide a differential analysis of each of them based onthe combined use of the LDS-751 (Exciton) DNA-dye, the thiazol orange(TO, Molecular Probes, Inc) RNA-dye, a fluorescently labeled anti-CD45monoclonal antibody, forward light scatter (FSC) and sideward lightscatter (SSC). This approach allowed the specific identification ofnucleated red cells, erythrocytes, reticulocytes, platelets,lymphocytes, monocytes, neutrophilic granulocytes, basophilicgranulocytes, eosinophilic granulocytes, and precursors of all nucleatedhematopoietic cells. However, the described multiparameter analysiscould not specifically differentiate between normal, reactive,regenerating and neoplastic cells coexisting in the same sample, neithercould this procedure further characterize these groups of cells. Afurther refinement of this patent was described later on in U.S. Pat. No6,287,791 by Terstappen and Chen, but they did not show any improvementin characterizing the different populations of leukocytes.

More recently, Orfao et al. described in US. Pat. No. 7,332,295 aprocedure for the multidimensional leukocyte differential analysis ofPB, BM and other body fluids, which specifically allowed identificationof dendritic cells and their subsets in addition to nucleated red cells,lymphocytes, monocytes, neutrophilic granulocytes, basophilicgranulocytes, eosinophilic granulocytes and hematopoietic precursors ofall nucleated cells. Furthermore, in U.S. Pat. No 5,538,855, Orfao etal. described a procedure that allowed a more detailed analysis of thelymphoid compartments through the simultaneous identification of up to12 different subsets of T, B and NK-cells in PB, BM and lymph nodesamples. In the patent, Orfao et al. used a combined staining for theCD3, CD19, CD56 (and/or CD16), CD4 and CD8 antigens in a 3-color singlestaining, where pairs of monoclonal antibodies conjugated with the samefluorochrome were used. Nevertheless, through this approach they couldneither further characterize the identified cell subsets nordiscriminate between normal and neoplastic cells; furthermore, somerelevant subsets of lymphoid and non-lymphoid cells (e.g. the TCRγδ+T-cells) present in a normal PB, BM or other tissues and body fluids,could not be specifically detected.

None of the referred procedures allowed directly for a more detailedcharacterization of the cells identified, including discriminationbetween normal, reactive, regenerating, and neoplastic/clonal cellpopulations. Such characterization and discrimination requires the useof a greater number of stainings associated with distinguishablefluorescence emissions and of flow cytometer instruments capable ofmeasuring a higher number of different fluorescence emissions.

Many other publications show that, based on specific immunophenotypicprofiles, normal cells can be discriminated from their neoplasticcounterparts, and that the discrimination capabilities (sensitivity fordiscriminating normal from clonal/malignant cells) increase with thenumber of fluorochrome-conjugated antibody reagents used to stain thesame sample. Some reports also show that normal and reactive cells aswell as maturing cells during BM regeneration, may show distinctimmunophenotypic profiles, but these cells could not be clearlydiscriminated from malignant cells. Importantly, panels of antibodycombinations that have been proposed thus far can not efficientlydiscriminate between normal, reactive, regenerating cell populations andtheir neoplastic counterparts in a systematic way and, at the same time,allow for a clear-cut discrimination between different diagnosticdisease categories through an extended immunophenotypic characterizationof the malignant cells.

With the introduction of the new generation of multi-laser flowcytometers in diagnostic laboratories, the potential space for buildingdifferent panels even with the same list of antibodies, hasexponentially increased due to the possibility of using a larger numberof different fluorescent compounds and simultaneously measure a highernumber of fluorescence emissions in single cells. In U.S. Pat. No.7,321,843, Orfao, Pedreira and Sobral da Costa propose a new approachbased on mathematical calculation procedures (e.g. the closest neighbourprinciple), where flow cytometry list mode data files containinginformation for each single cell measured about an unlimited number ofparameters could be generated, after several aliquots of a samplestained with different, partially overlapping, combinations ofmonoclonal antibodies had been measured in a flow cytometer. The utilityof this procedure could only be maximised with the increased multicolourcapabilities of the most recent flow cytometers. This is because theseprocedures require the design of efficient panels of combinations ofmonoclonal antibody reagents where common backbone markers are combinedwith other additional markers which vary for each combination ofmonoclonal antibodies in a panel. In parallel, the same authors alsodescribed (U.S. Pat. No. 7,507,548) a procedure for comparison of flowcytometry data from a case against a reference data file which wouldallow direct comparison of e.g. normal vs. neoplastic cells. However, inboth patents, the authors failed to propose a comprehensive antibodypanel that could be thoroughly and systematically applied to thediagnosis, classification, staging and/or monitoring of leukaemia,lymphoma and plasma cell dyscrasias.

Thus, existing protocols do not teach about how to specifically combinethe widely recommended monoclonal antibodies in a panel; they proposedifferent panels for the diagnostic classification and monitoring ofminimal disease; they focus in single, uniform disease groups; they showa limited efficiency once widely tested; they do not provide a way fordirect combination of the information measured for several aliquots froma single sample; and/or they have failed in systematicallydistinguishing normal/reactive versus clonal/neoplastic cells based ontheir immunophenotypic properties, in specific diseases such as chronicT and NK lymphoproliferative disorders (e.g. in the differentialdiagnoses of large granular lymphocytic leukemia).

The present inventors recognized these difficulties, and set out todesign improved, well-defined antibody panels that avoidmisinterpretation and over-interpretation of results. After carefulselection of the relevant markers, design of appropriate combinations ofantibodies in multi-color tubes, and the selection of suitedfluorochromes (based on need for brightness, compensation, stability,etc.), a set of antibody reagents was developed. The studies werecomplemented with extensive multicentric evaluation of the consensuspanels in order to reshape and achieve an optimal efficiency.

Accordingly, the invention provides in one embodiment a reagentcomposition for flow cytometric immunophenotyping of leukocytescomprising at least eight distinct fluorochrome-conjugated antibodiescomprising a set of at least three identification antibodies for theidentification of a leukocyte population of interest and at least fourcharacterization antibodies for further characterization and/orclassification of said leukocyte population. A reagent composition asprovided herein comprises a panel of antibodies directed against one ofthe following combinations of markers: (a) CD20, CD4, CD45, CD19, Igλ,CD8, Igx, CD56, TCRγδ, CD3 and CD38, wherein the antibody within eitherone of the pairs CD20/CD4, Igλ/CD8 and CD19 /TCRγδ is conjugated to thesame fluorochrome (LST reagent composition); (b) CD20, CD45, CD8, Igλ,CD56, Igκ, CD4, CD3, CD14 and CD38, wherein the antibody within eitherone of the pairs CD8/Igλ, CD56/Igκ and CD3/CD14 is conjugated to thesame fluorochrome (SST reagent composition); (c) CD45, CD138, CD38,CD56, β2micro, CD19, cyIgκ and cyIgλ (PCST reagent composition) or (d)cyCD3, CD45, cyMPO, cyCD79a, CD34, CD19, CD7 and CD3 (ALOT reagentcomposition).

In a preferred embodiment, the composition comprises monoclonalantibodies. CD stands for cluster designation and is a nomenclature forthe identification of specific cell surface antigens defined bymonoclonal antibodies. Abbreviations used are as follows:cyIgκ=cytoplasmic IgG kappa chain; cyIgλ=cytoplasmic IgG lambda chain;β2micro=β₂ microglobulin; cyMPO=cytoplasmic myeloperoxidase.(Monoclonal) antibodies against the indicated markers can becommercially obtained from various companies, including Becton/Dickinson(BD) Biosciences, Dako, Beckman Coulter, CYTOGNOS, Caltag, Pharmingen,Exbio, Sanquin, Invitrogen, and the like.

Suitable fluorochromes for conjugating antibodies are known in the art.As will be understood, the fluorochromes used within a reagentcomposition should be distinguishable by flow cytometry. Thefluorochromes are preferably selected for brightness, limited spectraloverlap and limited need for compensation, stability, etc. The followingpanel of fluorochromes is of particular use in a reagent compositionaccording to the invention: (1) pacific blue (PacB) or Horizon V450, (2)pacific orange (PacO) or AMCA, (3) fluorescein isothiocyanate (FITC) orAlexa488, (4) phycoerythrin (PE), (5) peridinin chlorophylprotein/cyanine 5.5 (PerCP-Cy5.5), PerCP or PE-TexasRed, (6)phycoerythrin/cyanine7 (PE-Cy7), (7) allophycocyanine (APC) or Alexa647,and (8) allophycocyanine/H7 (APC-H7), APC-Cy7, Alexa680 or Alexa700.After multiple testing rounds, the present inventors observed that verygood results can be obtained if the following fluorochromes are chosen:Pacific Blue or Horizon V450, Pacific Orange, fluorescein isothiocyanate(FITC), phycoerythrin (PE), peridinin chlorophyl protein/cyanine 5.5(PerCp-Cy5.5), PE-Cy7, allophycocyanine (APC), and APC-H7.

The expression “wherein the antibody within either one of the pairs isconjugated to the same fluorochrome” is meant to indicate that bothantibodies of the first pair are conjugated to fluorochrome A and thatboth antibodies of the second pair are conjugated to fluorochrome B.Thus, within each pair the fluorochromes arc the same but betweendifferent pairs the fluorochromes are distinguishable.

Each of the reagent compositions can be used as such, e.g. for thescreening of a lymphoid disease. See FIG. 2 for a schematic overview ofexemplary applications of the various reagent compositions. Theinvention thus also relates to diagnostic kits comprising one or morereagent compositions. However, the compositions are also advantageouslyused in combination with one or more further reagent compositions, inparticular reagent compositions designed for the further screening andclassification of the disease. The expression “in combination with” doesnot refer to the physical combination or mixing of the reagentcompositions, but to their application in separate (consecutive)analysis steps and combination of the data thus obtained. For example, ascreening tube used in combination with a characterization tube involvestwo separate analytical steps on separate aliquots of the samebiological sample, each using one of the reagent compositions, followedby data recording and evaluation.

Therefore, the invention also relates to a set of at least two reagentcompositions, said set comprising a reagent composition as describedherein above, and at least one further reagent composition comprisingdistinct fluorochrome-conjugated antibodies. Thus, both reagentcompositions comprise a distinct panel of antibodies, although someantibodies might be present in both compositions. It is very convenientif the panel of distinct fluorochromes is essentially the same for eachof the reagent compositions, and that up to eight differentfluorochromes are used in total.

In one embodiment, a set of at least two reagent compositions comprisesa reagent composition according to (a) and/or (b) and/or (c) definedherein above (i.e. LST reagent and/or SST reagent and/or PCST reagent),together with at least one further reagent composition comprisingdistinct fluorochrome-conjugated antibodies, preferably directed againstone of the following combinations of markers:

-   -   (i) CD20, CD45, CD23, CD10, CD79b, CD19, CD200 and CD43;    -   (ii) CD20, CD45, CD31, LAIR1, CD11c, CD19, IgM and CD81    -   (iii) CD20, CD45, CD103, CD95, CD22, CD19, CXCR5 and CD49d    -   (iv) CD20, CD45, CD62L, CD39, HLADR, CD19, CD27 and CD31    -   (v) CD4, CD45, CD7, CD26, CD3, CD2, CD28 and CD8    -   (vi) CD4, CD45, CD27, CCR7, CD3, CD45RO, CD45RA and CD8    -   (vii) CD4, CD45, CD5, CD25, CD3, CD11c and CD8    -   (viii) CD4, CD45, CD57, CD30, CD3, CD11c and CD8    -   (ix) CD4, CD45, cyPerforin, cyGranzyme, CD3, CD16, CD94 and CD8    -   (x) CD4, CD45, CD279, CD3 and CD8    -   (xi) CD2, CD45, CD7, CD26, CD3, CD56, CD5 and CD19    -   (xii) CD16, CD45, CD57, CD25, CD3, CD56, CD11c and CD19    -   (xiii) HLADR, CD45, cyPerforin, cyGranzyme, CD3, CD56, CD94 and        CD19; or    -   (xiv) CD45, CD138, CD38, CD28, CD27, CD19, CD117 and CD81

In one embodiment, a set comprises both the LST and SST reagents and atleast one of the above further reagents. In another embodiment, a setcomprises at least the PCST reagent.

In another embodiment, a set of at least two reagent compositionscomprising a first reagent composition according to (d) as definedherein above (ALOT reagent), and at least one further reagentcomposition comprising distinct fluorochrome-conjugated antibodiesdirected against one of the following combinations of markers:

-   -   (i) CD20, CD45, CD58, CD66c, CD34, CD19, CD10 and CD38    -   (ii) IgK, CD45, cyIgμ, CD33, CD34, CD19, IgM, CD117 and Igλ,        wherein the antibodies against IgM and CD117 are conjugated to        the same fluorochrome    -   (iii) CD9, CD45, terminal deoxynucleotidyl transferase (TdT),        CD13, CD34, CD19, CD22 and CD24    -   (iv) CD21, CD45, CD15, CDw65, NG2, CD34, CD19, CD123 and CD81,        wherein the antibodies against CD15 and CDw65 are conjugated to        the same fluorochrome    -   (v) cyCD3, CD45, TdT, CD99, CD10, CD1a and CD3    -   (vi) cyCD3, CD45, CD2, CD117, CD4, CD8, CD7 and CD3    -   (vii) cyCD3, CD45, TCRγδ, TCRαβ, CD33, CD56, cyTCRβ and CD3    -   (viii) cyCD3, CD45, CD44, HLADR, CD45RA, CD123 and CD3    -   (ix) HLADR, CD45, CD16, CD13, CD34, CD117, CD11b and CD10    -   (x) HLADR, CD45, CD35, CD64, CD34, CD117, immune receptor        expressed by myeloid cell 2 (IREM2) and CD14    -   (xi) HLADR, CD45, CD36, CD105, CD34, CD117, CD33 and CD71    -   (xii) HLADR, CD45, CD15, chondroitin sulfate proteoglycan (NG2),        CD34, CD117, CD7 and CD38    -   (xiii) HLADR, CD45, CD42a, CD61, CD203c, CD34, CD117, CD123 and        CD4, wherein the antibodies against CD42a and CD61 are        conjugated to the same fluorochrome    -   (xiv) HLADR, CD45, CD41, CD25, CD34, CD117, CD42b and CD9    -   (xv) HLADR, CD45, CD41, CD25, CD34, CD117, CD42b and CD9; or    -   (xvi) HLADR, CD45, TdT, CD56, CD34, CD117, CD22 and CD19.

Exemplary sets of reagent compositions include at least one reagentcomposition as recited in Tables 1 and/or 6 herein below, together withat least one reagent composition as recited in any one of Tables 2 -5and 7-9.

A further aspect relates to a diagnostic kit for flow cytometricimmunophenotyping of leukocytes, wherein the kit comprises one or moresets of at least two reagent compositions described above. The kit mayin addition comprise other useful components, such as instructions foruse, sample preparation reagent, buffer, and/or control samples.

Reagent compositions, sets and diagnostic kits provided herein findtheir application in various fields. For example, the proposed panelscan be applied as a whole or only partially depending on the nature ofthe sample, medical indication or the specific goal. The panel may usespecific monoclonal antibody reagents by a single or several differentmanufacturers, in combination with different cell preparation techniquesfor staining of cell surface only and/or cell surface plus intracellularmarkers. Similarly, the reagent compositions may be upgraded into apanel including combinations containing monoclonal antibody reagentsconjugated with >8 different fluorochromes where the same backbonemarkers are maintained and combined with additional or similar markers.An antibody composition may also act as a core panel to which newcombinations are added with different goals, including monitoring of theimmune system.

In one embodiment, there is provided a diagnostic kit for theidentification and characterization of mature lymphoid cells, comprisingthe SST and/or LST and/or PCST reagent composition together with atleast one reagent composition for detecting B-cell chroniclymphoproliferative disorder (B-CLPD) comprising antibodies againstCD20, CD45, CD23, CD10, CD79b, CD19, CD200 and CD43; CD20, CD45, CD31,LAIR1, CD11c, CD19, IgM and CD81; CD20, CD45, CD103, CD95, CD22, CD19,CXCR5 and CD49d; or CD20, CD45, CD62L, CD39, HLADR, CD19, CD27 and CD31.

For example, there is provided a diagnostic kit for the identificationand characterization of mature lymphoid cells, comprising the SST and/orLST and/or the PCST reagent composition together with at least onereagent composition for detecting T-cell chronic lymphoproliferativedisorder (T-CLPD) comprising antibodies against CD4, CD45, CD7, CD26,CD3, CD2, CD28 and CD8; CD4, CD45, CD27, CCR7, CD3, CD45RO, CD45RA andCD8; CD4, CD45, CDS, CD25, CD3, CD11e and CD8; CD4, CD45, CD57, CD30,CD3, CD11c and CD8; CD4, CD45, cyPerforin, cyGranzyme, CD3, CD16, CD94and CD8; or CD4, CD45, CD279, CD3 and CD8.

Another exemplary diagnostic kit for the identification andcharacterization of mature lymphoid cells comprises the SST and/or LSTand/or PCST reagent composition together with at least one reagentcomposition for detecting NK-cell chronic lymphoproliferative disorder(NK-CLPD) comprising antibodies against CD2, CD45, CD7, CD26, CD3, CD56,CD5 and CD19; CD16, CD45, CD57, CD25, CD3, CD56, CD11c and CD19; HLADR,CD45, cyPerforin, cyGranzyme, CD3, CD56, CD94 and CD19; CD45, CD138,CD38, CD56, β2micro, CD19, cyIgκ and cyIgλ; or CD45, CD138, CD38, CD28,CD27, CD19, CD117 and CD81.

In yet another embodiment, there is provided a diagnostic kit for theidentification and characterization of mature lymphoid cells, comprisingthe SST and/or LST and/or PCST reagent composition together with atleast one reagent composition for detecting plasma cell dyscrasias (PCD)comprising antibodies against CD45, CD138, CD38, CD56, β2micro, CD19,cyIgκ and cyIgλ; or CD45, CD138, CD38, CD28, CD27, CD19, CD117 and CD81.

Also provided is a diagnostic kit for identification andcharacterization of immature lymphoid cells, comprising the ALOT reagentcomposition together with at least one reagent composition for detectingB-cell precursor ALL (BCP-ALL) comprising antibodies against CD20, CD45,CD58, CD66c, CD34, CD19, CD10 and CD38; Igκ, CD45, cyIgμ, CD33, CD34,CD19, IgM, CD117 and Igλ, wherein the antibodies against IgM and CD117arc conjugated to the same fluorochrome; CD9, CD45, TdT, CD13, CD34,CD19, CD22 and CD24; or CD21, CD45, CD15, CDw65, NG2, CD34, CD19, CD123and CD81, wherein the antibodies against CD15 and CDw65 are conjugatedto the same fluorochrome.

In a further aspect, a diagnostic kit for identification andcharacterization of immature lymphoid cells comprises the ALOT reagentcomposition together with at least one reagent composition for detectingT-cell precursor ALL (T-ALL) comprising antibodies against cyCD3, CD45,TdT, CD99, CD10, CD1a and CD3; cyCD3, CD45, CD2, CD117, CD4, CD8, CD7and CD3; cyCD3, CD45, TCRγδ, TCRαβ, CD33, CD56, TCR =F1 and CD3; orcyCD3, CD45, CD44, HLADR, CD45RA, CD123 and CD3.

In still a further aspect, a diagnostic kit is provided foridentification and characterization of myeloid cells, comprising theALOT reagent composition together with at least one reagent compositionfor detecting acute myeloid leukemia (AML), myelodysplastic syndrome(MDS)/chronic myeloproliferative disorder (MPD), comprising antibodiesagainst HLADR, CD45, CD16, CD13, CD34, CD117, CD11b and CD10; HLADR,CD45, CD35, CD64, CD34, CD117, IREM2 and CD14; HLADR, CD45, CD36, CD105,CD34, CD117, CD33 and CD71; HLADR, CD45, CD15, NG2, CD34, CD117, CD7 andCD38; HLADR, CD45, CD42a, CD61, CD203c, CD34, CD117, CD123 and CD4,wherein the antibodies against CD42a and CD61 are conjugated to the samefluorochrome; HLADR, CD45, CD41, CD25, CD34, CD117, CD42b and CD9;HLADR, CD45, CD41, CD25, CD34, CD117, CD42b and CD9; or HLADR, CD45,TdT, CD56, CD34, CD117, CD22 and CD19.

The invention also relates to a method for flow cytometricimmunophenotyping of leukocytes, comprising the steps of providing abiological sample comprising leukocytes and contacting at least aportion (aliquot) of the sample with a reagent composition providedherein. Any type of (human) sample known or suspected to containleukocytes may be used. For example, the sample is peripheral blood,bone marrow, tissue sample such as lymph nodes, adenoid, spleen, orliver, or other type of body fluid such as cerebrospinal fluid, vitreousfluid, synovial fluid, pleural effusions or ascitis. Preferably, themethod comprises contacting a first aliquot of said sample with a firstreagent composition of a set according to the invention and contactingat least a second aliquot of said sample with a further reagentcomposition of said set; analyzing leukocytes in said aliquots in a flowcytometer; and storing and evaluating the data obtained. Typically, step(c) comprises the use of software for data integration andmultidimensional analysis of flow cytometry files. Very suitable for usein a method of the invention is the software commercially available fromCYTOGNOS SL (Salamanca, Spain) under the tradename INFINICYT™. TheINFINICYT™ software can automatically combine the immunophenotypicinformation of the selected cell populations from multiple tubesaccording to the so-called nearest neighbor calculations in whichindividual cells from one aliquot of a sample are matched withcorresponding individual cells from another aliquot of the same sample,according to their backbone markers and scatter profile. The INFINICYTprocedure can transform the herein presented 8-color EuroFlow panelsinto 12, 16, or ≥20-color immunostainings, dependent on the number oftubes per panel and the number of backbone markers per tube. Theantibody panels and the INFINICYT software can be used in combinationwith all currently available flow cytometers that allow 8-colorimmunostainings, such as FACSCanto™ II, FACSAria, LSRII, DAKOCyAn™,Gallio, etc.

LEGEND TO THE FIGURES

FIG. 1. Composition of three Categories of antibody panels

FIG. 2. Diagnostic flow diagram showing potential applications of theEuroFlow antibody panels.

DETAILED DESCRIPTION

1. Introduction

The present study was performed by the The EuroFlow Consortium(LSHB-CT-2006-018708) who initiated the project “Flow Cytometry for Fastand Sensitive Diagnosis and Follow-up of Haematological Malignancies”,which includes the design of standardized multicolor immunophenotypingprotocols for diagnosis, classification, and monitoring of leukemias,lymphomas and plasma cell dyscrasias. A key innovative component ofthese protocols are the EuroFlow panels of antibody combinations(EuroFlow panels). This EuroFlow invention relates among others topanels of combinations of antibody reagents (reagent compositions),which can be used to define normal, reactive, regenerating and malignanthematopoietic cells in a standardized way. As such, they allow for acomprehensive immunophenotypic diagnosis, classification, staging andmonitoring of both chronic and acute leukemias, myelodysplasticsyndromes, myeloproliferative disorders, mastocytosis, paroxysmalnocturnal haemoglobinuria, lymphomas and plasma cell dyscrasias. For thefirst time, the proposed panels are not based on subjective expertopinions, but they have been tested prospectively and modified forimproving the answer to the most frequent medical indications of flowcytometry immunophenotyping. In turn, these panels are designed in aninnovative way to be applied in combination with both conventional dataanalysis approaches and new interactive and semi-automated data analysisprocedures in which information on single cells is combined for allparameters derived from the measurement of staining a sample with theantibody panel.

In order to establish these panels, the following sequential steps wereperformed:

1. Evaluation of all relevant markers used in the field for theirusefulness or added value

-   -   Markers proposed by European networks, such as ELN, EMN, ERIC;    -   Markers proposed by the 2006 Bethesda International Consensus in        US;    -   New markers for mature B-cell malignancies as proposed by        Rawstron et al.

2. Design and selection of combinations of markers (≥6), which canrecognize normal vs. reactive vs. regenerating vs. abnormal/malignantcells within a specific compartment of hematopoietic cells (celllineage, differentiation pathway, maturation stage and/or functionalsubset).

3. Evaluation of the proposed panels of combinations of antibodyreagents in primary samples from healthy subjects and patients.

4. Repeated testing and optimization of the combinations of antibodyreagents (choice of marker, choice of antibody clone, and choice offluorochrome) based on uncommitted goals and room for improvement.

5. Evaluation of the optimized versions of the EuroFlow panels on largeseries of well-defined patient samples and samples from healthycontrols.

Compared with pre-existing knowledge, this is the first design of acomprehensive panel (after prospectively evaluated in a multicentricway), which allows both discrimination between normal, reactive,regenerating and clonal/neoplastic cells and classification, staging andmonitoring of clonal/neoplastic haematopoietic disorders, providing aclear indication about: 1) those markers required to be stained incommon for appropriate and reproducible identification of all cellpopulations of interest in all stained aliquots of a sample, and 2) howthey should be combined with further characterization markers inspecific combinations of fluorochrome-conjugated antibody reagents. Inaddition, information about the goals of each combination is also givenas indication about when and how to apply it. The invention was madeonly after extensive antibody panel testing and several redesigningcycles.

The EuroFlow panels of reagents are composed of subsets of one ormultiple combinations (named tubes) of antibodies conjugated with eightor more fluorescent compounds, each of said combinations of reagentshaving different goals. The EuroFlow panels consist of three differentcategories: Category 1, Category 2 and Category 3 antibody panels (FIG.1).

2. Categories of Antibody Panels

The following purposes are pursued by the EuroFlow panels of antibodycombinations:

The Category 1 antibody panels aim at identification andcharacterization of different subsets of mature lymphoid cells,including normal, reactive, regenerating and neoplastic B-, T-, NK-cellsand plasma cells, particularly in samples where a clonal and/or neoplastic lymphoid disorder is suspected because of e.g. lymphocytosis, lymphnode enlargement, splenomegaly, monoclonal serum component, unexplainedneurological symptoms, etc. The Category 2 antibody panels aim atidentification and characterization of normal, reactive, regeneratingand neoplastic immature (or early maturing) T- and/or B-lymphoid cells,particularly in samples suspected of containing neoplastic lymphoidprecursors. The Category 3 antibody panels aim at identification andcharacterization of normal, reactive, regenerating and neoplasticimmature, maturing and matured myeloid cells, particularly in samplessuspected of containing neoplastic myeloid cells or neoplastic cellsexpressing myeloid-associated markers.

Within each of said three Categories, some of the combinations ofantibody reagents are aimed to be used in a single-tube screening stepwith more broad aims, while others are more likely applied in multi-tubeclassification steps when more specific target populations have alreadybeen identified in the screening step.

2.1 Category 1 Antibody Panels

The Category 1 antibody panels are composed of three screening tubesaimed at the initial identification and characterization of the specificsubgroups of mature lymphoid cells present in samples which containnormal or high cell counts (e.g. normal peripheral blood) and low cellcounts (e.g. vitreous humor), respectively, and four different sets ofmulti-tube antibody combinations aiming at further characterization ofB-, T-, NK-cells and plasma cells. The Category 1 screening tubes may beused to screen for the presence of clonal and neoplas tic T, B- orNK-cells and plasma cells in samples with relatively high and low cellcounts, respectively. Typical examples of low cell count samples arefine needle aspirates (FNA), cerebrospinal fluid (CSF), and vitreoushumor.

The above mentioned screening tubes are now termed as lymphoid screeningtube for high cell count samples (abbreviated as LST), small sample tube(SST) for low cell count samples, and a separate plasma cell screeningtube (PCST). In turn, the other four sets of tubes are devoted, amongother uses, to further characterize the identified clonal or neoplasticB-, T-, NK-cells and plasma cells in patients with different B-, T- andNK-cell chronic lymphoproliferative disorders (abbreviated as BCLPD,TCLPD and NKCLPD, respectively) and plasma cell dyscrasias (abbreviatedas PCD), respectively.

2.2 Category 2 Antibody Panels

The Category 2 antibody panels consist of a screening tube devoted tothe initial identification and characterization of immature lymphoid vs.non-lymphoid precursors, and two sets of tubes for further detailedcharacterization of B-cell and T-cell precursors. Among otherapplications, the screening tube may be used for the classification ofacute leukemias into lymphoid vs. non-lymphoid versus undifferentiatedversus biphenotypic or bilineage acute leukemias. This tube might alsobe used for the discrimination between normal/regenerating andneoplastic/malignant B-cell precursors in the bone marrow, peripheralblood and other tissues and T-cell precursors in the thymus,respectively. Consequently, this screening tube is now termed as acuteleukemia orientation tube (abbreviated ALOT). The other two sets oftubes may be used, among other applications, for the characterization oflymphoid blast cells showing B-cell and T-cell commitment in patientswith B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and T-ALLas well as in patients with acute leukemia showing an undifferentiatedor biphenotypic/bilineage phenotype.

2.3 Category 3 Antibody Panels

Finally, the Category 3 antibody panels are composed of a screening tubewhich aims at discriminating between lymphoid and non-lymphoid precursorcells and which is identical to the screening tube described above inCategory 2 (ALOT tube), and one additional set of tubes aiming at thecharacterization of maturing non-lymphoid cells from the earliesthematopoietic precursors to mature myeloid cells, including thedifferent maturation stages of cells committed to the erythroid,megakaryocytic, monocytic, neutrophilic, eosinophilic, basophilic, mastcell and dendritic cell lineages as well as the more immature,uncommitted hematopoietic precursors and stromal cells in case they arepresent in the sample (e.g. in BM). Among other uses, this set of tubesmay be applied for the characterization of neoplastic/clonal diseasesand other disorders in which the cells of interest (e.g.:clonal/neoplastic, altered/aberrant cells) display myeloiddifferentiation such as in patients with acute myeloid leukemia (AML),myelodysplastic syndrome (MDS), chronic myeloproliferative disorder(MPD), paroxysmal nocturnal haemoglobinuria (PNH), mastocytosis,idiopatic cytopenia of undetermined significance (ICUS)- and/or acuteleukemia with myeloid-associated markers as it occurs in biphenotypicand bilineage acute leukemias. This group of tubes is abbreviated fromnow on as AML/MDS/MPD.

2.4 Types of Markers Used in EuroFlow Panels

In each tube of the EuroFlow panels, two types of antibody reagents arecombined: 1) reagents which mainly aim at identifying the precise cellpopulations of interest present in the sample (Backbone markers), whichalso provide additional information about the phenotypic characteristicsof said cell populations, and; 2) reagents mainly devoted to furthercharacterization/classification of said cell populations as well asother groups of cells in the sample (Characterization markers).

Typically, backbone markers are repeated in each tube of the followingsets of tubes: AML/MDS/MPD, BCP-ALL, T-ALL, B-CLPD, T-CLPD, NK-CLPD,PCD). In addition, backbone markers of the B-CLPD set of tubes are alsocommon to the LST and SST screening tubes, backbone markers of the PCDset of tubes are common to the PCST, and backbone markers of the BCP-ALLand the T-ALL sets of tubes are also common to the ALOT; finally, two ofthe backbone markers in the AML panel (i.e..: CD34 and CD45) are alsocommon to the ALOT tube.

In turn, characterization markers are combined in a comprehensive way ineach tube, so that they would allow distinguishing normal, reactiveregenerating vs clonal/neoplastic cells, even when present in lownumbers and in case of neoplastic cells they would allow furtherdiagnosis, subclassification, staging and monitoring of acute andchronic leukemias, lymphomas and plasma cell dyscrasias. For thispurpose in multi-tube panels, each tube is devoted to a specific aimrelated to full characterization and monitoring of a disease entity ifcombined with the information from the corresponding screening tube(e.g. for the diagnosis, staging and monitoring of CLL, tube#4 of theB-CLPD multi-tube panel in combination with the LST will suffice) or forspecific disease—associated information (e.g. tube# 24 in the BCP-ALLmulti-tube panel shown in Table 7 is specifically devoted fordistinguishing between normal/reactive and regenerating versus malignantB-cell precursors and minimal residual disease monitoring in BCP-ALL).

2.5 INFINICYT Software Tools for Optimal Application of the EuroFlowPanels

The antibody panels according to the invention can be used incombination with the INFINICYT software tools which are commerciallyavailable. The software is based on recently described procedures forgenerating files with an unlimited number of parameters through mergingdata files and calculating the information derived from the measurementof markers in one sample aliquot to the individual cells measured inother aliquots of the same sample, using different combinations ofantibody reagents which only have partial overlap (U.S. Pat. No.7,321,843), as well as for comparisons between different samples ordifferent groups of samples (U.S. Pat. No. 7,507,548).

3. Composition of the Category 1 Antibody Panels for Mature LymphoidCells

In the multi-tube combinations of antibody reagents of the Category 1antibody panels for mature lymphoid cells the selected common backbonemarkers vary between the four different panels and consist of (antibodyCD plus fluorochrome compound number): 1) CD20-1, CD45-2 and CD19-6 forB-CLPD; 2) CD4-1, CD45-2, CD3-5 and CD8-8 for T-CLPD; 3) CD45-2, CD3-5,CD56-6 and CD19-8 for NK-CLPD, and; 4) CD45-1, CD138-2, CD38-3 andCD19-6 for PCD. In addition, the backbone markers used in the B-CLPDtube are also used in the LST and SST screening tubes, and the backbonemarkers of the PCD tubes are also used in the PCST.

The backbone markers in each of these multi-tube combinations ofantibody reagents aim at providing delineation of the groups of cells ofinterest and the specific identification of neoplastic cells indiagnostic and follow-up samples containing enough numbers of tumorcells. In addition, for each of the four multi-tube combinations,backbone markers are combined with a variable number of additionalcharacterization markers which further contribute to the discriminationbetween normal, reactive, regenerating and neoplastic/clonal B-, T,NK-cells and plasma cells, respectively, even when these are present inminimal numbers (e.g. minimal residual disease monitoring and diseasestaging), as well as for the distinction between clonal/neoplastic cellsfrom different disease categories.

3.1. The Lymphoid Screening Tube (LST), the Small Sample Tube (SST), andthe Plasma Cell Screening Tube (PCST)

The EuroFlow LST reagent composition was designed and approved forevaluation of several suspected clinical conditions, such aslymphocytosis, lymph node enlargement, splenomegaly, monoclonal serumcomponents, unexplained neurological symptoms, unexplained cytopenias,etc. (FIG. 2). The composition of an exemplary LST tube is provided inTable 1. This tube detects aberrant mature lymphocyte populations of B,T and NK lineage. However, this 8-color tube does not allow the precisediagnosis and classification of the detected aberrant lymphocytepopulations. This typically needs further characterization with theB-CLPD, T-CLPD, NK-CLPD and/or PCD tubes (see Sections 3.2, 3.3 and3.4).

The EuroFlow SST reagent composition is a modified version of EuroFlowLST, specially designed for evaluation of small samples and samples with(very) low cell counts, such as fine needle aspirates (FNA),cerebrospinal fluid (CSF), vitreous humor, etc. For this special aim,the tube allows the unequivocal recognition of normal leukocytes presentin these samples, e.g. B, T, NK cells and monocytes as well as anycoexisting aberrant cell population. The composition of an exemplary SSTreagent is provided in Table 1.

The EuroFlow PCST reagent composition is specially designed forscreening of plasma cells in order to detect aberrancies or clonality.In case of aberrant or clonal plasma cells, complementary phenotypiccharacterization is achieved via the 2-tube PCD antibody panel.

In any of the Tables of the present application, fluorochrome number 1corresponds to pacific blue (PacB) or Horizon V450, number 2 to pacificorange (PacO) or AMCA, number 3 to fluorescein isothiocyanate (FITC) orA1exa488, number 4 to phycoerythrin (PE), number 5 to peridininchlorophyl protein/cyanine 5.5 (PerCP-Cy5.5), PerCP or PE-Texas Red,number 6 to phycoerythrin/cyanine7 (PE-Cy7), number 7 toallophycocyanine (APC) or Alexa647, and number 8 to allophycocyanine/H7(APC-H7), APC-Cy, Alexa680 or Alexa700. A preferred combination ispacific blue (PacB), pacific orange (PacO), fluorescein isothiocyanate(FITC), phycoerythrin (PE), peridinin chlorophyl protein/cyanine 5.5(PerCP-Cy5.5), phycoerythrin/cyanine7 (PE-Cy7), allophycocyanine (APC),and allophycocyanine/H7 (APC-H7).

TABLE 1 Single tube EuroFlow screening combinations for normal andmalignant/aberrant mature lymphoid cells Fluorochrome Tube# 1 2 3 4 5 67 8 1 (LST) CD20^(BB) CD45^(BB) Igλ Igκ CD5 CD19^(BB) CD3 CD38 and andand and CD4 CD8 CD56 TCRγ6 2 (SST) CD20^(BB) CD45^(BB) CD8 CD56 CD4CD19^(BB) CD3 CD38 and and and Igλ Igκ CD14 3 (PCST) CD45^(BB)CD138^(BB) CD38^(BB) CD56 β2micro CD19^(BB) CyIgκ CyIgλ ^(BB)Thesemarkers act as Backbone markers when LST or SST results are combinedwith the results obtained with multi-tube EuroFlow classificationcombination for B-CLPD (see Table 2) or when the PCST results arecombined with the results obtained with the 2-tube EuroFlowclassification combination for PCD (see Table 5); LST = lymphoidscreening tube; SST = small sample tube; PCST = plasma cell screeningtube.

3.2. The Multi-Tube Antibody Panel for B-Cell ChronicLymphoproliferative Disorders (B-CLPD)

The B-CLPD is designed to classify mature B-cell malignancies accordingto WHO entities based on flow cytometric data only (see Table 2).Information obtained using the LST simultaneously or sequentially has tobe integrated into the B-CLPB panel (e.g. via INFINICYT software). TheB-CLPD panel is designed to work in cases in which the malignant B cellpopulation can be purified to >90% using the backbone markers CD20,CD19, and CD45, regardless of the cell material analyzed.

The panel is designed to work in a modular way, i.e. it is not necessaryto stain the whole panel if the pre-test probability for a particularB-cell malignancy is high. In those instances the panel will allow todiagnose a particular entity using a reduced number of tubes. Forexample, the LST tube#1 plus tube# 5 are sufficient to diagnose CLL witha very high positive predictive value (PPV).

TABLE 2 Multi-tube EuroFlow classification combinations for B-cellchronic lymphoproliferative disorders (B-CLPD) Tube# 1 2 3 4 5 6 7 8 Aim4 CD20^(BB) CD45^(BB) Igλ Igκ CD5 CD19^(BB) CD3 CD38 LST: Detection ofand and and and (almost) all mature B-cell CD4 CD8 CD56 TCRγ6malignancies 5 CD20^(BB) CD45^(BB) CD23 CD10 CD79b CD19^(BB) CD200 CD43Identification, of all CLL cases 6 CD20^(BB) CD45^(BB) CD31 LAIR1 CD11cCD19^(BB) IgM CD81 Identification of all HCL cases; characterization ofbenign B-, T-, NK-cells 7 CD20^(BB) CD45^(BB) CD103 CD95 CD22 CD19^(BB)CXCR5 CD49d Identification of DLBCL, FL, MZL LPL 8 CD20^(BB) CD45^(BB)CD62L CD39 HLADK CD19^(BB) CD27 CD31 Identification of DLBCL, FL, MZL,LPL ^(BB)Backbone marker; Tube 4 is identical to the LST (see Table 1).The described tubes can also be successfully applied for disease stagingand monitoring.

3.3. The Multi-Tube Antibody Panel for T-Cell ChronicLymphoproliferative Disorders (T-CLPD)

The EuroFlow T-CLPD aims for diagnosis and classification of matureT-cell malignancies. Also for this panel, information obtained with theLST simultaneously and sequentially is preferably integrated with T-CLPDtubes (e.g, via harmonization with the INFINICYT software). The panel isdesigned to work in cases in which the malignant T cell population canbe purified to >90% using the backbone-markers CD3, CD4, CD8, and CD45,regardless of the cell material analyzed. The combination of LST andT-CLPD tubes can detect T-cell malignancies of both TCRαβ and TCRγδlineages.

TABLE 3 Multi-tube EuroFlow classification combinations for T-cellchronic lymphoproliferative disorders (T-CLPD) Tube# 1 2 3 4 5 6 7 8 Aim 9 CD4^(BB) CD45^(BB) CD7 CD26 CD3^(BB) CD2 CD28 CD8^(BB) Phenotypiccharacterization; Identification of Sezary syndrome 10 CD4^(BB)CD45^(BB) CD27 CCR7 CD3^(BB) CD45RO CD45RA CD8^(BB) Phenotypiccharacterization; Assessment of maturation stage 11 CD4^(BB) CD45^(BB)CD5 CD25 CD3^(BB) HLADR cyTCL1 CD8^(BB) Phenotypic characterization;Identification of T-PLL 12 CD4^(BB) CD45^(BB) CD57 CD30 CD3^(BB) CD11cCD8^(BB) Phenotypic characterization; Cytotoxic phenotype andidentification of anaplastic lymphoma 13 CD4^(BB) CD45^(BB) cyPerforincyGranzyme CD3^(BB) CD16 CD94 CD8^(BB) Phenotypic characterization;Assessment of cytotoxic- associated phenotypes; Identification of T-LGL14 CD4^(BB) CD45^(BB) CD279 CD3^(BB) CD8^(BB) Identification oflymphomas derived from follicular helper T cells (angioimmunoblastic Tcell lymphomas) ^(BB)Backbone marker; the tubes can also be successfullyapplied for disease staging and monitoring.

3.4. The Multi-Tube Antibody Panel for NK-Cell ChronicLymphoproliferative Disorders (NK-CLPD)

The EuroFlow NK-CLPD tube aims at the discrimination between aberrantand normal/reactive NK-cells. The NK-CLPD panel uses four Backbonemarkers: CD45-2, CD3-5, CD56-6 and CD19-8. (NK-CLPD)

TABLE 4 Multi-tube EuroFlow classification combinations for NK-cellchronic lymphoproliferative disorders (NK-CLPD) Tube# 1 2 3 4 5 6 7 8Aim; MRD 15 CD2 CD45^(BB) CD7 CD26 CD3^(BB) CD56^(BB) CD5 CD19^(BB)Detection of aberrant NK cell phenotype 16 CD16 CD45^(BB) CD57 CD25CD3^(BB) CD56^(BB) CD11c CD19^(BB) Detection of aberrant NK cellphenotype 17 HLADR CD45^(BB) cyPerforin cyGranzyme CD3^(BB) CD56^(BB)CD94 CD19^(BB) Detection of aberrant NK cell phenotype; Assessment ofcytotoxic effector phenotype ^(BB)Backbone marker; the tubes can also besuccessfully applied for disease staging and monitoring.

3.5. The Multi-Tube Antibody Panel for Plasma Cell Dyscrasias (PCD)

The EuroFlow PCD panel comprises two tubes with four Backbone markers:CD45-1, CD138-2, CD38-3 and CD19-6 for PCD. (Table 5); tube #18 isidentical to the PCST (tube #3) in Table 1. The PCD panel aims at theidentification and enumeration of plasma cells as well as at thediscrimination between normal polyclonal plasma cells such as inreactive plasmacytosis versus aberrant monoclonal plasma cells such asin monoclonal gammopathies of undetermined significance (MGUS),smoldering and symptomatic multiple myeloma (MM), plasma cell leukemias(PCL), amyloidosis, and extramedullary plasmacytoma, and thedifferential diagnosis among these plasma cell dyscrasias. Incombination with the EuroFlow LST and B-CLPD panels, this multi-tubeantibody panel will also contribute to the diagnosis of other plasmacell dyscrasias such as Waldenstrom's macroglobulinemia andlymphoplasmacytic lymphoma (LPL).

TABLE 5 Multi-tube EuroFlow classification combinations for plasma celldyscrasias (PCD) Tube# 1 2 3 4 5 6 7 8 Aim; 18 CD45^(BB) CD138^(BB)CD38^(BB) CD56 β2micro CD19^(BB) cyIgκ cyIgλ PCST: Detection of aberrantand clonal plasma cells 19 CD45^(BB) CD138^(BB) CD38^(BB) CD28 CD27CD19^(BB) CD117 CD81 Complementary phenotypic characterization andevaluation of markers with potential prognostic impact ^(BB)Backbonemarker. The described tubes can also be successfully applied for diseasestaging and monitoring; Tube #18 is identical to the PCST (Tube #3 inTable 1).

4. Composition of the Category 2 Panel for Immature Lymphoid Cells

4.1. The Acute Leukemia Orientation Tube (ALOT)

The EuroFlow ALOT tube was designed for assessment of the nature ofimmature blast cell populations in acute leukemia patients (B, T versusnon-lymphoid, undifferentiated or mixed phenotype acute leukemias) andconsequent orientation towards the most appropriate complementaryantibody panel(s): BCP-ALL, T-ALL, and/or AML/MDS/MPD. The compositionof the ALOT tube is provided in Table 6; the markers CD45-2, CD34-5 andCD19-6 act as backbone markers when the information of the ALOT iscombined with the BCP-ALL panel (e.g.: using the INFINICYT software) andthe cyCD3-1, CD45-2 and CD3-8 markers act also as common backbonemarkers when the information of the ALOT is combined with the T-ALLpanel; in addition CD45-2 and CD34-5 are also common backbone markers tothe AML/MDS/MPD multi-tube panel.

TABLE 6 Single tube EuroFlow combination for lineage assignment of blastcells in acute leukemias Tube# 1 2 3 4 5 6 7 8 20 cyCD3^(BB) CD45^(BB)cyMPO cyCD79a CD34^(BB) CD19^(BB) CD7 CD3^(BB) (ALOT) ^(BB)These markersact as Backbone markers when results are combined with the resultsobtained with multi-tube EuroFlow classification combinations forBCP-ALL or T-ALL (see Table 7 and Table 8, respectively);

The ALOT is not suitable for exclusion of a hematological malignancy,because the ALOT antibody combination is not sufficient for thatpurpose. However, when the ALOT is combined with the LST and 4 tubes ofthe AML/MDS protocol (tube # 29, 30, 31 and 32), virtually all types ofhematological malignancies can be detected (not classified) or excluded(FIG. 2).

4.2. The Multi-Tube Antibody Panel for B-Cell Precursor AcuteLymphoblastic Leukemia (BCP-ALL)

The EuroFlow BCP-ALL tube aims at the recognition and classification ofall classically defined BCP-ALL (pro-B-ALL, common-ALL, pre-B-ALL) oralternative BCP-ALL classifications, including immunophenotypicclassifications associated with well-defined molecular aberrations, suchas specific fusion genes. The information obtained with the BCP-ALL tubeset needs to be combined with ALOT, based on the backbone markersCD45-2, CD34-5 and CD19-6 and using appropriate data analysis software(e.g. via the INFINICYT software) (Table 7).

TABLE 7 Multi-tube EuroFlow classification combinations for B-cellprecursor ALL (BCP-ALL) Tube# 1 2 3 4 5 6 7 8 Aim 21 CD20 CD45^(BB) CD58CD66c CD34^(BB) CD19^(BB) CD10 CD38 Diagnosis and classification ofBCP-ALL; Detection of LAP markers 22 Igκ CD45^(BB) CyIgμ CD33 CD34^(BB)CD19^(BB) IgM Igλ Diagnosis and and classification of BCP-ALL; CD117Detection of phenotypes assiciated with molecular aberrations 23 CD9CD45^(BB) TdT CD13 CD34^(BB) CD19^(BB) CD22 CD24 Diagnosis andclassification of BCP-ALL; Detection of phenotypes assiciated withmolecular aberrations; Detection of LAP markers 24 CD21 CD45^(BB) CD15NG2 CD34^(BB) CD19^(BB) CD123 CD81 Subclassification of BCP- and ALL;Detection of LAP CDw65 markers ^(BB)Backbone markers. The tubes can alsobe successfully applied for disease staging and monitoring.

4.3. The Multi-Tube Antibody Panel for T-Cell Acute LymphoblasticLeukemia (T-ALL)

The EuroFlow T-ALL panel consists of four tubes and uses cyCD3-1,CD45-2, and CD3-8 as backbone markers (see Table 8) in common with theALOT. The T-ALL panel aims at the recognition and classification of allclassically defined T-ALL (immature T-ALL, common thymocytic T-ALL,mature T-ALL) or alternative T-ALL classification, e.g. based on TCRprotein expression (cyTCRβ, TCRαβ, TCRγδ) or based on association withwell-defined molecular aberrations.

TABLE 8 Multi-tube EuroFlow classification combinations for T-ALL Tube#1 2 3 4 5 6 7 8 Aim 25 cyCD3^(BB) CD45^(BB) TdT CD99 CD5 CD10 CD1aCD3^(BB) Diagnosis of T-ALL; Identification of LAP markers 26 cyCD3^(BB)CD45^(BB) CD2 CD117 CD4 CD8 CD7 CD3^(BB) Diagnosis of T-ALL,classification of T-ALL, and determine the maturation stage of arrest;Identification of LAP markers 27 cyCD3^(BB) CD45^(BB) TCRγδ TCRαβ CD33CD56 Cy TCRβ CD3^(BB) Diagnosis of T-ALL and determine the maturationstage of arrest; Identification of LAP markers 28 cyCD3^(BB) CD45^(BB)CD44 CD13 HLADR CD45RA CD123 CD3^(BB) Subclassification of T-ALL^(BB)Backbone markers. The tubes can also be successfully applied fordisease staging and monitoring.

5. Composition of the Category 3 Panel for Myeloid Cells

The EuroFlow AML/MDS/MPD panel comprises two complementary markercombinations (tubes 29-32 and tubes 32-35; see Table 9), all of themcontaining HLA-DR-1, CD45-2, CD34-5 and CD117-6 as backbone markers(Table 9). The first set of tubes (tubes 29-32) is designed topreferably be used in combination with EuroFlow ALOT in order to aim atthe detection and classification (lineage assignment and maturation) ofmyeloid malignancies, such as in AML and MDS, with a major focus onimmature neutrophilic lineage (tube 30), monocytic lineage (tube 31),and erythroid lineage (tube 32). These four tubes also contribute to thedetection of APL (tube 32), PNH (tubes 29 and 30) and other aberrantmyeloid phenotypes.

The second set of tubes (tubes 33-35) provides additional informationabout megakaryocytic, basophilic, plasmacytoid dendritic lineages (tube33), as well as relevant information for the diagnosis of mastocytosisin association (or not) with AML/MDS (tube 34). Tube 35 furthercharacterizes AML/MDS, and it is particularly focused on the aberrantexpression of lymphoid-associated markers and abnormal lymphoidmaturation.

TABLE 9 Multi-tube EuroFlow classification combinations for AML/MDS/MPDTube# 1 2 3 4 5 6 7 8 Aim 29 HLADR^(BB) CD45^(BB) CD16 CD13 CD34^(BB)CD117^(BB) CD11b CD10 Diagnosis of AML 30 HLADR^(BB) CD45^(BB) CD35 CD64CD34^(BB) CD117^(BB) IREM2 CD14 Diagnosis and subclassification of AMLand PNH especially focussed on neutrophilic lineage 31 HLADR^(BB)CD45^(BB) CD36 CD105 CD34^(BB) CD117^(BB) CD33 CD71 Diagnosis andsubclassification of AML especially focussed on monocytic lineage 32HLADR^(BB) CD45^(BB) CD15 NG2 CD34^(BB) CD117^(BB) CD7 CD38 Diagnosisand subclassification of AML and APL especially focussed on erythroidlineage 33 HLADR^(BB) CD45^(BB) CD42a CD203c CD34^(BB) CD117^(BB) CD123CD4 Diagnosis and plus subclassification of AML CD61 especially focussedon megakaryocytic, basophilic, and plasmacytoid dendritc lineages 34HLADR^(BB) CD45^(BB) CD41 CD25 CD34^(BB) CD117^(BB) CD42b CD9Characterization of AML/MDS 35 HLADR^(BB) CD45^(BB) TdT CD56 CD34^(BB)CD117^(BB) CD22 CD19 Characterization of AML/MDS ^(BB)Backbone markers;This multi-tube set of antibody combinations is not only useful for AML,MDS and MPD detection, but also for mastocytosis, PNH, ICUS. The tubescan also be successfully applied for disease staging and monitoring.

REFERENCES

1.—Consensual European Immunophenotyping panels for leukemia.www.leukemia-net.org/content/home/.

2.—Wood B L, Arroz M, Barnett D, DiGiuseppe J, Greig B, Kussick S J,Oldaker T, Shenkin M, Stone E, Wallace P. 2006 Bethesda InternationalConsensus recommendations on the immunophenotypic analysis ofhematolymphoid neoplasia by flow cytometry: optimal reagents andreporting for the flow cytometric diagnosis of hematopoietic neoplasia.Cytometry B Clin Cytom. 2007;72 Suppl 1:S14-22.

3.—Bone M C, Castoldi G, Knapp W, Ludwig W D, Matutes E, Orfao A, van'tVeer M B. Proposals for the immunological classification of acuteleukemias. European Group for the Immunological Characterization ofLeukemias (EGIL). Leukemia. 1995;9:1783-6

4.—Rawstron A C, Orfao A, Beksac M, Bezdickova L, Brooimans R A, BumbeaH, Dalva K, Fuhler G, Gratama J, Hose D, Kovarova L, Lioznov M, Mateo G,Morilla R, Mylin A K, Omedé P, Pellat-Deceunynck C, Perez Andres M,Petrucci M, Ruggeri M, Rymkiewicz G, Schmitz A, Schreder M, Seynaeve C,Spacek M, de Tute R M, Van Valckenborgh E, Weston-Bell N, Owen R G, SanMiguel JF, Sonneveld P, Johnsen H E; European Myeloma Network. Report ofthe European Myeloma Network on multiparametric flow cytometry inmultiple myeloma and related disorders. Haematologica. 2008;93:431-8.

5.—Rawstron A C, Villamor N, Ritgen M, Böttcher S, Ghia P, Zehnder J L,Lozanski G, Colomer D, Moreno C, Geuna M, Evans P A, Natkunam Y, CoutreS E, Avery E D, Rassenti L Z, Kipps T J, Caligaris-Cappio F, Kneba M,Byrd J C, Hallek M J, Montserrat E, Hillmen P. Internationalstandardized approach for flow cytometric residual disease monitoring inchronic lymphocytic leukaemia. Leukemia. 2007;21:956-64.

6.—Matutes E, Owusu-Ankomah K, Morilla R, Garcia Marco J, Houlihan A,Que T H, Catovsky D. The immunological profile of B-cell disorders andproposal of a scoring system for the diagnosis of CLL. Leukemia.1994;8:1640-5.

1.-13. (canceled)
 14. A reagent composition for flow cytometric immunophenotyping of leukocytes and characterization of normal and aberrant subpopulations thereof, wherein the reagent composition comprises fluorochrome-conjugated antibodies directed against the following combination of markers: cyCD3, CD45, cyMPO, cyCD79a, CD34, CD19, CD7, and CD3, wherein the antibody against cyCD3, the antibody against CD45, the antibody against cyMPO, the antibody against cyCD79a, the antibody against CD34, the antibody against CD19, the antibody against CD7, and the antibody against CD3 are each conjugated to a fluorochrome that has a different fluorescence emission from the other fluorochromes.
 15. A set of at least two reagent compositions, said set comprising a first reagent composition according to claim 14, and at least one further reagent composition comprising distinct fluorochrome-conjugated antibodies directed against one of the following combinations of markers: (i) CD20, CD45, CD58, CD66c, CD34, CD19, CD10, and CD38; (ii) Igκ, CD45, cyIgμ, CD33, CD34, CD19, IgM, CD117, and Igλ, wherein the antibodies against IgM and CD117 are both conjugated to the same fluorochrome; (iii) CD9, CD45, TdT, CD13, CD34, CD19, CD22, and CD24; (iv) CD21, CD45, CD15, CDw65, NG2, CD34, CD19, CD123, and CD81, wherein the antibodies against CD15 and CDw65 are both conjugated to the same fluorochrome; (v) cyCD3, CD45, TdT, CD99, CDS, CD10, CD1a, and CD3; (vi) cyCD3, CD45, CD2, CD117, CD4, CD8, CD7, and CD3; (vii) cyCD3, CD45, TCRγδ, TCRαβ, CD33, CD56, cyTCRβ, and CD3; (viii) cyCD3, CD45, CD44, CD13, HLADR, CD45RA, CD123, and CD3; (ix) HLADR, CD45, CD16, CD13, CD34, CD117, CD11b, and CD10; (x) HLADR, CD45, CD35, CD64, CD34, CD117, IREM2, and CD14; (xi) HLADR, CD45, CD36, CD105, CD34, CD117, CD33, and CD71; (xii) HLADR, CD45, CD15, NG2, CD34, CD117, CD7, and CD38; (xiii) HLADR, CD45, CD42a, CD61, CD203c, CD34, CD117, CD123, and CD4, wherein the antibodies against CD42a and CD61 are both conjugated to the same fluorochrome; (xiv) HLADR, CD45, CD41, CD25, CD34, CD117, CD42b, and CD9; or (xv) HLADR, CD45, TdT, CD56, CD34, CD117, CD22, and CD19.
 16. The reagent composition or set of reagent compositions according to claim 14 or 15, wherein each reagent composition comprises antibodies conjugated to PacB or Horizon V450, PacO or AMCA, FITC or Alexa488, PE, PerCP-Cy5.5, PerCP or PE-TexasRed, PE-Cy7, APC or Alexa647, and APC-H7, APC-Cy7, Alexa680 or Alexa700.
 17. The set of reagent compositions according to claim 16, comprising at least the reagent composition as recited in tube #20 of Table 6, together with at least one reagent composition as recited in any one of Tables 7-9.
 18. The set of reagent compositions according to claim 16 or 17, comprising at least the reagent composition as recited in tube #20 of Table 6, the reagent composition as recited in tube #1 of Table 1, and the reagent compositions as recited in tubes #29, 30, 31 and 32 of Table
 9. 19. A diagnostic kit for flow cytometric immunophenotyping of leukocytes and characterization of normal and aberrant subpopulations thereof, comprising a set of at least two reagent compositions according to claim 15, optionally together with instructions for use, buffer, and/or control samples.
 20. The diagnostic kit according to claim 19 for identification and characterization of myeloid cells, comprising a set of reagent compositions recited in claim 15 under (ix) through (xv).
 21. A method for flow cytometric immunophenotyping of leukocytes and characterization of normal and aberrant subpopulations thereof, comprising the steps of providing a biological sample comprising leukocytes; contacting a first aliquot of said sample with a first reagent composition of a set according to claim 15 and contacting at least a second aliquot of said sample with a further reagent composition of said set; analyzing leukocytes in said aliquots in a flow cytometer; and storing and evaluating the flow cytometric data obtained to characterize the leukocytes for the presence of normal and aberrant subpopulations.
 22. The method according to claim 21, wherein said sample is peripheral blood, bone marrow, tissue sample, or a body fluid.
 23. The method according to claim 21, wherein step (c) comprises the use of software for data integration and multidimensional analysis of flow cytometry files.
 24. The method according to claim 23, wherein the software is INFINICYT™. 